Human immunodeficiency virus type 1 (HIV-1) has been identified as the causative agent of AIDS (Barre-Sinoussi et al., 1983 Science 220:868-871; reviewed in Fauci (1988) Science 239:617-622). Currently, the World Health Organization estimates that between 13 and 14 million people are infected with HIV worldwide, of which one million are in the United States. The virus attacks the body's immune system and is thought to be fatal in 90% of the patients who have had AIDS for two or more years.
HIV-1 is a complex retrovirus whose life cycle is characterized by distinct kinetic phases. The key viral mediator of emergence from the initial phase, in which only non-structural proteins are produced, is the virus-encoded regulatory protein Rev, which regulates the cytoplasmic accumulation of virion genomic and structural mRNAs. Arrigo et al., 1989 J. Virol. 63: 4875-4881; Emerman et al., 1989 Cell 57: 1155-1165; Felber et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 1495-1499; Malim et al. 1989 Nature 338: 254-257) Kim et al., 1989 J. Virol. 63: 3708-3713; reviewed in Cullen and Greene (1989) Cell 58:423-426.
Rev is a 20-kDa protein, and is highly charged and phosphorylated. Hauber et al., 1988 J. Virol 62:4801-4804; Cochrane et al., 1989 Virology 171:264-266. It is a nuclear protein (Cullen et al. 1988 J. Virol. 62:2498-2501; Felber et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:1495-1499; Perkims et al. 1989 J. AIDS 2:256-263) and accumulates in the nucleolus (Malim et al., (1989) Cell 58:205-214; Cochrane et al. 1990 J. Virol. 64:881-885).
The genome of HIV is complex and contains at least nine open reading frames. Different proteins are expressed by the production of alternatively spliced RNAs from the full-length precursor RNA. In the nucleus of the host cell, Rev binds to an HIV RNA known as the RRE (Rev-responsive element) and exports singly spliced and unspliced viral mRNAs, which contain the RRE, to the cytoplasm where they are translated into protein (Emerman et al., 1989 Cell 57: 1155-1165; Felber et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:1495-1499; Malim et al., 1989 Nature 338: 254-257). Rev is essential for viral replication in human cells (Sodroski et al., (1986) Nature 321:412-417; Sadaie et al., (1988) Science 239:910-914; Terwillinger et al., 1988 J. Virol. 62: 655-658; Cullen and Greene (1989) Cell 58:423-426), which makes Rev an attractive target for the development of therapeutics against HIV infections and for the treatment of AIDS (Baltimore (1988) Nature 335:395-396). Preventing Rev function will prevent export and therefore translation of viral mRNAs into protein and will inhibit viral replication.
The Rev-RRE interaction, although necessary, is not sufficient for the activation of RRE-containing mRNAs. Additional cellular factor(s) interacting with Rev are necessary for Rev function (Malim et al., (1989) Cell 58:205-214; Olsen et al., (1990) Genes Dev. 4:1357-1364; Ahmed et al., 1990 Genes Dev. 4:1014-1022; Benko et al., 1990 New Biol. 2: 1111-1122). Rev mutants have been created that bind RRE but fail to function (Malim et al., (1989) Cell 58:205-214; Olsen et al., (1990) Genes Dev. 4:1357-1364). These carboxy-terminal mutants exhibit a trans-dominant negative phenotype in tissue culture cells (Malim et al., (1989) Cell 58:205-214; Venkatesh and Chinnadurai, (1990) Virology 178:327-330). Inferred from this finding is the existence of a cellular factor or factors that is necessary to effect Rev function. Several cellular proteins have been identified that bind to or interact with Rev protein, and which have been proposed to be vital for Rev function: the mammalian nucleolar protein B23 (Fankhauser et al.,(1991) Mol. Cell. Biol. 11:2567-2575) YL2 (Luo et al., (1994) J. Virol. 68:3850-3856) and eIF-5A (Ruhl et al., (1993) 123:1309-1320).
Identifying the cellular factor essential for Rev function provides an important opportunity for identifying a new class of antiviral drugs. Identifying a therapeutic drug that inhibits Rev cofactor function provides an excellent opportunity for combination therapy. Because of the ability of HIV to mutate rapidly, experts agree that the best approach to combating the disease lies in simultaneous treatment with two or more therapies that target different components of the virus. There are only three approved drugs to treat HIV and AIDS, AZT, DDC, and DDI, none of which provides an effective cure, and which all work by blocking a viral enzyme called reverse transcriptase. Drugs that inhibit Rev function are likely to have toxicities distinct from those of reverse transcriptase inhibitors, and as a result will complement or act in synergy with other therapies.
Development of an effective method and composition for treatment of HIV infections is a critical goal of the pharmaceutical industry. The pharmaceutical industry has made numerous efforts to identify effective HIV drugs, with only limited success to date. It would be of great value to identify a new class of anti-HIV drug that blocks a viral or cellular target other than HIV reverse transcriptase. This target should lead to the development of a drug that is effective against viruses that are resistant to current therapy.
Development of an effective method of inhibiting the Rev effector is also likely to be useful for the treatment of other retroviruses containing Rev-like proteins such as HTLV-I and HTLV-II, HIV-1, HIV-2, HTLV-I and HTLV-II, although associated with different pathological processes, all require translation of incompletely spliced mRNA species for production of viral structural genes. Rev is conserved in all HIV and simian immunodeficiency virus isolates (Malim et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:8222-8226; Holland et al., (1990) J. Virol. 64:5966-5975). RREs from different primate immunodeficiency viruses can be functionally interchanged to some extent (Malim et al., (1989) Proc. Natl. Acad. Sci. U.S.A. 86:8222-8226). Similar to Rev, Rex-I is essential for virus replication (Rosenblatt et al., (1988) Science 240:916-919). Rex-I facilitates the cytoplasmic accumulation of unspliced or singly spliced viral mRNAs that encode Gag, Pol, and Env structural proteins (Hidaka et al. (1988) EMBO J. 7:519-523; Inoue et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3653-3657; Ohta et al., (1988) J. Virol. 62:4445-4451; Seiki et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 85:3618-3622). Rex-I and Rex-II proteins and the Rex.sub.I RE and Rex.sub.II RE are entirely interchangeable and Rex-II can replace Rev in HIV-I (Kim et al., (1991) 65:405-414). Rev, however, is unable to replace Rex-II in the HTLV-II system (Kim et al., (1991) 65:405-414).
It is likely that the same cellular factor(s) required for export of HIV-1, HIV-2, HTLV-I, and HTLV-II structural RNAs interacts with both the Rev proteins of HIV-1 and HIV-2 and the Rex proteins of HTLV-I and HTLV-II (Olsen et al., (1990) Genes Dev. 4:1357-1364). Transdominant repressors of Rex-I function are also transdominant suppressors of Rev function (Rimsky et al., (1989) Nature 335:738-740). Similarly, Rev inhibits Rex function in a dominant manner when the Rev responsive subregion of the RRE is deleted (Ahmed et al., (1990) Genes Dev. 4:1014-1022).
Drug development often relies on the screening of a large number of potential inhibitors before a specific lead compound inhibitor is found. Assays developed for such screens are complex and must mimic the physiological activity of the target protein. Thus, it is critical for the development of these screens to define the proteins involved in the targeted process and to have discovered a means of purifying the necessary components of the assay for use in the assay. In addition, it is useful to have clones for the protein components of the assay to facilitate the production of the components. Therefore, there is a need in the art to identify viral and cellular constituents, preferably polypeptides, that can serve as useful targets for drug intervention, and for methods and compositions for identifying useful anti-viral agents and treating viral infections.